Altered mechanism of the alkanesulfonate FMN reductase with the monooxygenase enzyme

BBRC Biochemical and Biophysical Research Communications 331 (2005) 1137–1145 www.elsevier.com/locate/ybbrc

Altered mechanism of the alkanesulfonate FMN reductase with the monooxygenase enzyme q Benlian Gao, Holly R. Ellis * Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849, USA Received 24 March 2005 Available online 19 April 2005

Abstract The two-component alkanesulfonate monooxygenase system from Escherichia coli is comprised of an FMN reductase (SsuE) and a monooxygenase enzyme (SsuD) that together catalyze the oxidation of alkanesulfonate to the corresponding aldehyde and sulfite products. To determine the effects of protein interactions on catalysis, the steady-state kinetic parameters for SsuE were determined in single-enzyme assays and in the presence of the monooxygenase enzyme and alkanesulfonate substrate. In single-enzyme kinetic assays, SsuE followed an ordered sequential mechanism, with NADPH as the first substrate to bind and NADP+ as the last product to dissociate. However, in the presence of SsuD and octanesulfonate the kinetic mechanism of SsuE is altered to a rapid equilibrium ordered mechanism, and the Km value for FMN is increased 10-fold. These results suggest that both the SsuD enzyme and alkanesulfonate substrate are required to ensure that the FMN reductase reaction proceeds to form the ternary complex with the subsequent generation of reduced flavin transfer. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Alkanesulfonate monooxygenase; FMN reductase; SsuE; SsuD; Steady-state kinetics

Many bacterial organisms rely on inorganic sulfur for the biosynthesis of sulfur-containing compounds. Inorganic sulfur is poorly represented in aerobic soil, therefore bacteria in soil environments must have alternative sources for obtaining this element. In Escherichia coli, the limitation of inorganic sulfate leads to the synthesis of specific proteins that are able to utilize alternate sulfur compounds for growth [1]. Two of these proteins include an FMN reductase (SsuE) and an FMNH2-utilizing monooxygenase (SsuD) that are involved in the conversion of alkanesulfonates to sulfites and aldehydes [2]. The SsuD enzyme directly catalyzes

q Abbreviations: IPTG, isopropyl-b-D-thiogalactoside; FRG, general flavin reductase, FRP, NADPH-preferring flavin reductase; SsuE, alkanesulfonate FMN reductase; SsuD, alkanesulfonate monooxygenase. * Corresponding author. Fax: +1 334 844 6959. E-mail address: [email protected] (H.R. Ellis).

0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.04.033

the desulfonation reaction and requires reduced flavin, provided by SsuE. In the overall reaction scheme, SsuE is proposed to catalyze the reduction of FMN directly by NADPH to form FMNH2 (Scheme 1) [2]. The reduced flavin is then transferred to the SsuD enzyme which converts the alkanesulfonate to aldehyde and sulfite in the presence of molecular oxygen. Reduced flavin transfer is a rather unique mechanistic model for flavin monooxygenase enzymes because flavin cofactors are typically bound to proteins through strong noncovalent or covalent interactions. Bacterial flavindependent monooxygenases that utilize reduced flavin as a substrate are continually emerging, and include proteins involved in the degradation of herbicides, the synthesis of antibiotics, desulfurization reactions, and bioluminescence [3–12]. All of these two-component enzyme systems rely on a flavin reductase and a monooxygenase enzyme for catalytic activity. Two groups of flavin reductases are associated with the

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B. Gao, H.R. Ellis / Biochemical and Biophysical Research Communications 331 (2005) 1137–1145

Scheme 1.

flavin-dependent monooxygenase family of enzymes. Class I enzymes are standard flavoproteins that show a distinct flavin spectrum as purified, whereas class II enzymes do not contain any flavin prosthetic group and cannot be defined as flavoproteins [13–15]. Several flavin reductases that belong to the class I enzymes have been identified that are associated with reduced flavin transfer to bacterial luciferase, nitrilotriacetate monooxygenase, EDTA monooxygenase, and pristinamycin IIA synthase [5,9,11,16–18]. Based on previous studies and our own observations, the SsuE enzyme is a class II flavin reductase that does not contain flavin as a tightly bound prosthetic group [2]. Other class II flavin reductase enzymes belong to the two-component enzyme families of chlorophenol 4-monooxygenase, valanimycin monooxygenase, and 4-hydroxyphenylacetate 3-monooxygenase [10,19,20]. A principal difference among the class II flavin reductases is their specificity for FMN or FAD. Reduced flavin transfer between the FMN reductase and monooxygenase component must be tightly controlled to prevent the generation of H2O2, superoxide, and hydroxyl radicals from the nonenzymatic reaction of reduced flavin with molecular oxygen. Direct flavin transfer from the flavin reductase to the monooxygenase enzyme through protein interactions would protect the flavin from oxidation. Protein interactions have been identified between Vibrio harveyi luciferase and NADPH-preferring flavin reductase (FRP) [21]. Fluorescent labeling studies showed that the monomeric form of FRP was able to form a complex with the luciferase enzyme. Additional evidence demonstrating the role of protein interactions in flavin transfer was shown through steady-state kinetic analysis with V. harveyi luciferase and FRP, and Vibrio fischeri luciferase with general flavin reductase (FRG) [22,23]. Conversely, there were no apparent protein interactions detected by size-exclusion chromatography and kinetic analysis between the FAD reductase and monooxygenase enzyme of the 4-hydroxyphenylacetate 3-monooxygenase system [24]. Flavin transfer for this enzyme system is said to occur through the diffusion of the reduced flavin to the monooxygenase enzyme due to the high affinity of the monooxygenase enzyme for FADH2 coupled with a high intracellular concentration of the monooxygenase enzyme. Although there are now a large number of two-component monooxygenase systems that belong to this family, the mechanism of flavin transfer for the majority of

these systems has not been well defined. We are interested in the independent kinetic mechanism of each enzyme and how the mechanism may be altered due to the interaction of the other enzyme component. In these studies, we have analyzed the FMN binding affinity and kinetic mechanism of the bisubstrate SsuE enzyme in the presence and absence of SsuD and the alkanesulfonate substrate. The results were obtained using a combination of fluorescence spectroscopy, bisubstrate steadystate kinetic analysis, and NADP+ product inhibition studies. The effect of the monooxygenase enzyme on the kinetic mechanism of the FMN reductases while solely monitoring flavin reduction has not been previously described for the flavin-dependent two-component enzyme family. The results described herein provide unique insights regarding the role SsuD plays in the mechanism of flavin transfer in the alkanesulfonate monooxygenase system.

Materials and methods Materials. FMN, NADPH, NADP+, potassium phosphate (monobasic anhydrous and dibasic anhydrous), sodium chloride, ampicillin, streptomycin sulfate, and lysozyme were from Sigma (St. Louis, MO). Isopropyl-b-D-thiogalactoside (IPTG), glycerol, and ammonium sulfate were purchased from Fisher Biotech (Pittsburgh, PA). Octanesulfonate was from Fluka (Milwaukee, WI). Standard buffer contains 25 mM potassium phosphate, pH 7.5, and 10% glycerol unless otherwise noted. Construction of expression vectors. The individual cloning of the alkanesulfonate monooxygenase genes into an expression vector was performed utilizing the Seamless Cloning System. The T7 RNA polymerase-dependent expression vector pET21a (Novagen, Madison, WI) was amplified using the primers (5 0 CAG TCA CTC TTC CCA TAT GTA TAT CTC CTT CTT) and (5 0 GCT TGC CTC TTC ACT CGA GCA CCA CCA CCA CCA), and included the Eam1104I restriction sites to produce NdeI and XhoI overhangs following digestion with the enzyme. The ssuE and ssuD genes were obtained from genomic DNA prepared from E. coli strain K12. The ssuE gene was PCR-amplified using the primers (5 0 ATA AGG CTC TTC TAT GCG TGT CAT CAC CCT G) and (5 0 CAG TTT CTC TTC GGA GTT ACG CAT GGG CAT T) which included the Eam1104I restriction sites and engineered to produce NdeI and XhoI overhangs following digestion by Eam1104I for ligation into the pET21A expression vector. The ssuD gene was amplified using the primers (5 0 AAG GAA CTC TTC TAT GAG TCT GAA TAT G) and (5 0 TGC CAT CCT TCT GAG TTA GCT TTG CGC CAC) which included the Eam1104I restriction sites and engineered to contain NdeI and XhoI overhangs following digestion by Eam1104I for ligation into the pET21a expression vector. DNA vectors containing representative clones were submitted for sequence analysis at Davis Sequencing (University of California, Davis). Expression and purification of the alkanesulfonate monooxygenase proteins. Cells from frozen stocks were isolated on LB-agar plates containing 100 lg/mL ampicillin (LB-Amp). A single colony of E. coli BL21(DE3) containing the appropriate expression plasmid was used to inoculate 5 mL LB-Amp media which were incubated 7 h at 37 °C. A 1% inoculum of the 5 mL culture was used to inoculate 100 mL LBAmp media which was incubated overnight at 37 °C and used to inoculate two 1 L flasks of LB-Amp media. When the A600 value reached 0.8–0.9 for SsuE and 0.4–0.5 for SsuD, the flasks were moved from 37 to 18 °C, and isopropyl-b-D-thiogalactoside (IPTG) was added

B. Gao, H.R. Ellis / Biochemical and Biophysical Research Communications 331 (2005) 1137–1145 to a final concentration of 0.4 mM. The incubation was continued for 6 h and cells were harvested by centrifugation at 5000g for 15 min and stored at
80 °C. For the purification of SsuE, cells from the 2 L growth were resuspended in 100 mL standard buffer containing 4 lg/mL lysozyme. Cell lysis was performed by sonication, followed by the addition of 1.5% streptomycin sulfate to precipitate nucleic acids. Ammonium sulfate precipitation was from 20% to 45%. The pelleted protein from the 45% ammonium sulfate precipitation was resuspended in 150 mL standard buffer with 20% ammonium sulfate and loaded onto the phenyl Sepharose column. After washing the column with 20% ammonium sulfate buffer, the protein was eluted with a linear gradient from 20% to 0% ammonium sulfate in standard buffer (300 mL total volume), followed by elution with standard buffer alone. Fractions containing large A280 values were pooled and loaded onto a macro-prep high Q column. After washing the column with standard buffer, the protein was eluted with a linear gradient from 0 to 300 mM NaCl in standard buffer. Fractions determined to be pure by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) were pooled, precipitated with 45% ammonium sulfate, and resuspended in standard buffer containing 100 mM NaCl. The resuspended protein pellet was pooled, dialyzed against standard buffer containing 100 mM NaCl, aliquoted, frozen, and stored at
80 °C until needed. The procedure for the purification of the SsuD protein was similar to the purification for SsuE with the following exceptions. Ammonium sulfate precipitation was from 30% to 60%. The pelleted protein from the 60% ammonium sulfate precipitation was resuspended in 50 mL standard buffer, dialyzed, and loaded onto a macro-prep high Q column. After washing the column with standard buffer, the protein was eluted with a linear gradient from 0 to 300 mM NaCl in standard buffer. Fractions containing large A280 values were pooled and 20% ammonium sulfate was added before loading onto a phenyl Sepharose column. After washing the column with 20% ammonium sulfate buffer, the protein was eluted with a linear gradient from 20% ammonium sulfate to 0% in standard buffer (300 mL total volume), followed by elution with standard buffer alone. Fractions determined to be pure by SDS– PAGE were pooled, precipitated with 60% ammonium sulfate, and resuspended in standard buffer containing 100 mM NaCl. The resuspended protein pellet was pooled, dialyzed against standard buffer containing 100 mM NaCl, aliquoted, frozen, and stored at
80 °C. Flavin binding. Flavin binding to SsuE and SsuD was monitored by spectrofluorometric titration with FMN. Spectra were recorded on a Perkin Elmer LS 55 luminescence spectrometer (Palo Alto, CA) with an excitation wavelength at 280 nm and emission measurements at 344 nm. Both excitation and emission slit widths were set at 5 and 2.5 nm for SsuE and SsuD, respectively. For the titration of SsuE with FMN, a 1.0 mL solution of 0.1 lM SsuE (calculated using a molar extinction coefficient of 20.3 mM
1 cm
1 at 280 nm) in standard buffer containing 100 mM NaCl was titrated with between 0.022 and 0.44 lM FMN (=12.2 mM
1 cm
1), and the fluorescence spectrum was recorded following a 2 min incubation after each addition of FMN. Titration of SsuD was performed with 0.5 lM SsuD (calculated using a molar extinction coefficient of 46.9 mM
1 cm
1 at 280 nm) in standard buffer containing 100 mM NaCl (1.0 mL total volume). The fluorescence spectrum was recorded following the addition of aliquots of FMN (5.2–98.6 lM) to SsuD. The bound FMN was determined by the following equation [24]: ½FMNbound

ðI 0
I c Þ ; ¼ ½SsuE ðI 0
I f Þ

ð1Þ

where [SsuE] represents the initial concentration of enzyme. I0 is the initial fluorescence intensity of SsuE prior to the addition of FMN, Ic is the fluorescence intensity of SsuE following each

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addition of FMN, and If is the final fluorescence intensity. The concentration of FMN bound ([FMN]bound, y) was plotted against the total FMN ([FMN]total, x) to obtain the dissociation constant (Kd) according to Eq. (2) where n is the binding capacity of SsuE [24]

y¼

ðK d þ x þ nÞ þ

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðK d þ x þ nÞ2
4xn 2

:

ð2Þ

Steady-state kinetic measurements. Enzymatic assays to measure the activity of the SsuE enzyme were performed by monitoring spectrophotometrically the decrease in 340 nm absorbance due to the oxidation of NADPH to NADP+ (e = 6.22 mM
1 cm
1). Reactions were initiated by the addition of SsuE (0.01 lM) enzyme to a reaction mixture (1 mL) containing between 3 and 60 lM NADPH and 0.01 and 0.06 lM FMN in standard buffer with 100 mM NaCl at 25 °C. The activity of the SsuE enzyme in the presence of 0.01 lM SsuD enzyme and 200 lM octanesulfonate substrate was performed similarly as described above. The octanesulfonate substrate was added prior to the initiation of the reaction with SsuE and SsuD. For each assay, the background rate due to the nonenzymatic oxidation of NADPH was subtracted from the initial velocities determined with SsuE. Inhibition studies of the SsuE protein with NADP+ were performed in a reaction mixture (1.0 mL) containing 0.01 lM SsuE, 0.1 lM FMN, 0–30 lM NADP+, and concentrations of NADPH between 20 and 100 lM. The rate was obtained by monitoring the decrease in 340 nm absorbance as described for the steady-state kinetic measurements. Data analysis. The data from the assays were fit with the Enzfitter program (Biosoft, Cambridge, UK) using three possible mechanisms (Eqs. (3)–(5)). Initial reaction rates of the SsuE protein in the presence or absence of the SsuD enzyme and alkanesulfonate substrate were fit to the following equations: sequential mechanism (Eq. (3)), ping-pong mechanism (Eq. (4)), and equilibrium ordered mechanism (Eq. (5)): m k cat AB ¼ ; e K a B þ K b A þ AB þ K ia K b

ð3Þ

m k cat AB ¼ ; e K a B þ K b A þ AB

ð4Þ

m k cat AB ¼ : e K a K b þ K b A þ AB

ð5Þ

For these equations Ka and Kb are the Km values for substrates A and B, respectively. A and B are the substrate concentrations. Kia is the dissociation constant for substrate A. kcat is the turnover number of the enzyme. The data from the steady-state inhibition studies were fit with the Enzfitter program to the three following inhibition equations: competitive (Eq. (6)), noncompetitive (Eq. (7)), and uncompetitive (Eq. (8)): m k cat A ¼ þ A; e K a ð1 þ I=K is Þ

ð6Þ

m k cat A ¼ ; e ½Að1 þ I=K is Þ þ ½Að1 þ I=K ii Þ

ð7Þ

m k cat A ¼ : e K a þ ½Að1 þ I=K ii Þ

ð8Þ

The Ka represents the Michaelis constant for substrate A. A and I are the substrate concentration and inhibitor concentration, respectively. Kis is the slope inhibition constant and Kii is the intercept inhibition constant. kcat is the turnover number of the enzyme.

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Results Protein expression and purification The ssuD and ssuE genes PCR-amplified from genomic E. coli K12 were cloned independently into the T7 RNA polymerase-dependent expression vector pET21a (Novagen), and the proteins expressed in E. coli BL21(DE3). Initial expression of each enzyme at 37 °C resulted in the location of the protein in inclusion bodies. The enzymes were expressed at 18 °C following induction with IPTG to obtain soluble protein, and the proteins were purified by a combination of ammonium sulfate fractionation, hydrophobicity, and anionic exchange chromatography. The amount of SsuE or SsuD protein obtained from a 2 L growth was between 30 and 50 mg. The mass of each protein determined by mass spectrometric analysis was 41,605 ± 2.6 Da and 21,253 ± 1.5 Da for SsuD and SsuE, respectively. Data from sedimentation equilibrium analyses using multiple concentrations of proteins were consistent with a tetrameric structure for SsuD and a dimeric structure for SsuE as previously reported in the initial characterization of this enzyme system [2].

Fig. 1. Fluorimetric titration of the SsuE enzyme with FMN. A 0.1 lM concentration of SsuE enzyme was titrated with between 0.022 and 0.44 lM FMN. Emission intensity measurements at 344 nm were measured using an excitation wavelength at 280 nm. The change in fluorescence of the SsuE enzyme following the addition of FMN was converted to the estimated concentration of bound FMN (Eq. (1)) and plotted against the total FMN concentration. The solid line represents the fit of the titration curve to Eq. (2).

Flavin binding The affinity of each alkanesulfonate monooxygenase enzyme for oxidized FMN was determined through fluorescence spectroscopy. To identify the affinity of the SsuE enzyme for FMN, aliquots of an FMN solution were added to SsuE and the decrease in the protein emission intensity at 344 nm due to flavin binding was monitored. The concentration of flavin bound to the SsuE enzyme was plotted against the total concentration of FMN added with each aliquot (Fig. 1). The average Kd value for FMN binding from two separate experiments was 0.015 ± 0.004 lM with one FMN binding site per SsuE monomer. Similar experiments were performed with oxidized FMN and SsuD monitoring the decrease in emission intensity at 340 nm. The average Kd value for FMN binding to SsuD was 10.2 ± 0.4 lM with a stoichiometry for FMN binding of 1.2 ± 0.1 (data not shown). Steady-state kinetic analysis of the SsuE enzyme The bisubstrate reaction mechanism of SsuE involves the reduction of FMN by the pyridine nucleotide NADPH. Steady-state kinetic studies were performed to investigate the kinetic mechanism of SsuE in the absence of SsuD. Varying concentrations of FMN at several fixed concentrations of NADPH gave an intersecting pattern to the left of the e/m axis (Fig. 2A). A second set of experiments was performed varying NADPH at fixed levels of FMN (Fig. 2B). The intersection point was again to the left of the

Fig. 2. Initial velocity patterns of the SsuE catalyzed reduction of FMN. The assays measured the oxidation of NADPH by the SsuE protein in the presence of FMN. (A) Assays were performed with 0.01 lM SsuE, 0.02–0.06 lM FMN, and varying fixed concentrations of NADPH: 10 lM (circles), 20 lM (squares), 40 lM (diamonds), and 60 lM (triangles). (B) Assays were performed with 0.01 lM SsuE, 3– 80 lM NADPH, and varying fixed concentrations of FMN: 0.01 lM (circles), 0.02 lM (squares), 0.04 lM (diamonds), and 0.06 lM (triangles). Each experiment was performed in triplicate, and the lines represent fits of the data with Enzfitter software to the model for a sequential mechanism (Eq. (3)).

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Table 1 Steady-state kinetic parameters for SsuE

kcat (min
1) KFMN (lM) KNADPH (lM) KiNADPH (lM)

SsuE

SsuE + SsuD and octanesulfonate

116.0 ± 6.3 0.016 ± 0.002 5.4 ± 0.9 3.9 ± 2.4

104.0 ± 0.5 0.13 ± 0.01 0.00 3.5 ± 0.1

e/m axis. Both sets of experiments were best fit to an equation for a sequential mechanism, however they did not distinguish between an ordered or random sequential mechanism. Steady-state kinetic parameters determined from the fit of the data to Eq. (3) gave Km values of 0.016 ± 0.002 and 5.4 ± 0.9 lM for FMN and NADPH, respectively. A Kia value for NADPH of 3.9 ± 2.4, and an apparent kcat value of 116.0 ± 6.3 min
1 were obtained (Table 1). Inhibition studies Inhibition studies on SsuE were performed with NADP+ as the inhibitor to discern between an ordered or random mechanism and to determine the order of substrate binding and product release. For these experiments the concentration of NADPH was varied at different concentrations of NADP+ and fixed nonsaturating levels of FMN. Double reciprocal plots of the data intersect on the e/m axis (Fig. 3), and the data were best fit to the equation for competitive inhibition (Eq. (6)) which gave a Ki value of 13.6 ± 6 lM for NADP+. In addition, there was no apparent inhibition with saturating levels of NADPH, varying concentrations of FMN, and different fixed concentrations of NADP+. These results indicate that high substrate concentrations were able to reverse the effects of the inhibitor leading to the formation of

Fig. 3. Inhibition of the SsuE enzyme with the NADP+ product with varied concentrations of NADPH. Assays were performed with 0.01 lM SsuE, 0.1 lM FMN, 20–100 lM NADPH, and varying fixed concentrations of NADP+: 0 lM (triangles), 10 lM (diamonds), 20 lM (squares), and 30 lM (circles). Each experiment was performed in triplicate, and the lines represent fits of the data with the Enzfitter software to the model for competitive inhibition (Eq. (6)).

Scheme 2.

the EÆNADPH complex. An ordered sequential mechanism for a bisubstrate system shows competitive inhibition when the varied substrate and product inhibitor are competing for the same form of the enzyme. For these experiments the NADPH substrate and NADP+ product are competing for the free enzyme. The results from the NADP+ inhibition experiments confirm that the mechanism of SsuE proceeds by an ordered sequential mechanism with NADPH binding first followed by FMN to form a ternary complex. Following reduction of the flavin, the reduced FMNH2 product is released first followed by NADP+ (Scheme 2). Steady-state kinetic mechanism of the SsuE enzyme in the presence of the SsuD enzyme and alkanesulfonate substrate Steady-state kinetic analysis was performed to determine if the kinetic parameters of SsuE were altered in the presence of SsuD. For these experiments either NADPH or FMN was held constant at saturating levels while varying the alternate SsuE substrate in the presence of varied stoichiometric ratios of SsuE to SsuD. The rate was determined by monitoring the decrease in 340 nm absorbance due to the oxidation of NADPH. Under these conditions there was no significant change in the steady-state kinetic parameters or mechanism of SsuE (data not shown). Although there was no significant change in the kinetic parameters of SsuE in the presence of SsuD only, we were interested in determining if the kinetic parameters and mechanism of the SsuE enzyme were altered with the addition of the SsuD enzyme and saturating alkanesulfonate substrate. Experiments were performed similar to those already described with the addition of octanesulfonate and a stoichiometric amount of SsuD enzyme relative to SsuE. The octanesulfonate substrate was previously shown to be a preferred substrate for the SsuD enzyme [2]. Double reciprocal plots of initial reaction rates against varying concentrations of FMN at several fixed concentrations of NADPH intersect on the e/m axis (Fig. 4A). Double reciprocal plots of reaction rates against NADPH concentration at different fixed concentrations of FMN intersect to the left of the e/m axis above the x-axis (Fig. 4B). These results were best fit to an equation for a rapid equilibrium ordered mechanism with NADPH binding first followed by FMN to form the ternary complex to give a Km value

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Km value for FMN is observed in the presence of SsuD and the octanesulfonate substrate.

Discussion

Fig. 4. Initial velocity patterns of the SsuE catalyzed reduction of FMN with the SsuD enzyme and octanesulfonate substrate. The assays measured the oxidation of NADPH with varying FMN by the SsuE enzyme in the presence of the SsuD enzyme and octanesulfonate substrate. (A) Assays were performed with 0.01 lM SsuE and SsuD, 200 lM octanesulfonate, 0.01–0.08 lM FMN, and varying fixed concentrations of NADPH: 10 lM (circles), 20 lM (squares), 40 lM (diamonds), and 60 lM (triangles). (B) Assays were performed with 0.01 lM SsuE, 0.01 lM SsuD, 200 lM octanesulfonate, 5–80 lM NADPH, and varying fixed concentrations of FMN: 0.02 lM (circles), 0.04 lM (squares), 0.06 lM (diamonds), and 0.1 lM (triangles). Each experiment was performed in triplicate, and the lines represent fits of the data with the Enzfitter software to the model for an equilibrium ordered mechanism (Eq. (5)).

for FMN of 0.13 lM ± 0.01, a Kia value of 3.5 ± 0.1 lM, and a kcat value of 104.0 ± 0.5 min
1 (Table 1). A rapid equilibrium ordered mechanism indicates that the Km value for the first substrate is appreciably lower than the Kia value, and can typically be confirmed by analyzing the data from Fig. 4A as a secondary plot of the slopes versus 1/[FMN] concentration [26]. For a bireactant equilibrium ordered mechanism a replot of the data should be linear and extrapolate through the origin. It was difficult to verify this mechanism further because FMN is inhibitory at high concentrations which limits the analysis of the slope effects. The inhibitory effect of increased flavin concentrations on the specific activity was previously observed with the alkanesulfonate monooxygenase system [2]. However, the data strongly suggest that the mechanism of the SsuE protein is modified from an ordered to a rapid equilibrium ordered kinetic mechanism and a 10-fold increase in the

Bacterial two-component monooxygenase systems that utilize reduced flavin as a substrate are continually being identified. Although these enzyme systems catalyze distinct reactions, a central theme in this family is the presence of a flavin-dependent reductase involved in flavin reduction followed by the transfer of reduced flavin to the monooxygenase component. The mechanism of flavin transfer could either occur through a diffusion mechanism or by direct flavin transfer involving protein interactions between the reductase and monooxygenase enzyme. The goal of these studies was to first establish the kinetic mechanism and parameters of SsuE in single-enzyme assays and then compare these results with the kinetic behavior of SsuE in the presence of the SsuD enzyme with and without the alkanesulfonate substrate. Initial studies focused on determining the affinity of each enzyme for oxidized FMN. These studies were central in establishing the specificity of each enzyme for oxidized flavin, and to determine if oxidized FMN binding to SsuD would interfere with further kinetic analysis with both SsuE and SsuD enzymes. The SsuE enzyme bound one FMN per monomer and had a 1000-fold lower Kd value than SsuD for oxidized flavin (Fig. 1). Based on these results the flavin reductase has a greater affinity for the oxidized flavin than the monooxygenase enzyme. Similar results were obtained for the flavin reductase in the two-component enzyme system involved in styrene degradation that also relies on flavin transfer between a class II flavin reductase and monooxygenase enzyme in the kinetic mechanism [25]. While specific for FAD, the flavin reductase enzyme was shown to have a 10-fold lower Kd value for FAD when compared to the monooxygenase enzyme. The higher affinity of the flavin reductase for the oxidized form of the flavin species in this two-component family appears to play an important role in promoting flavin specificity between the FMN reductase and monooxygenase enzyme. Data from the steady-state kinetic analysis of SsuE alone strongly support a sequential mechanism with the formation of a ternary complex before any chemistry occurs (Fig. 2). Formation of the ternary complex likely results in the transfer of electrons directly from NADPH to the flavin. Product inhibition studies with NADP+ further specify an ordered sequential mechanism with NADPH binding first followed by FMN as diagrammed in Scheme 2 (Fig. 3). Following reduction of the flavin, the reduced flavin product is released first and transferred either directly or by diffusion to SsuD. These re-

B. Gao, H.R. Ellis / Biochemical and Biophysical Research Communications 331 (2005) 1137–1145

sults seem to be inconsistent with binding assays which demonstrate the ability of FMN to bind without the pyridine nucleotide substrate. However, the kinetic mechanism does not suggest that the second substrate cannot bind without the first substrate, but dictates that the reaction can only occur if the enzyme follows an ordered mechanism. An ordered sequential kinetic mechanism is consistent with other characterized class II flavin reductases that utilize flavin as a substrate, and further establishes SsuE as a flavin reductase that lacks a bound flavin prosthetic group [13]. Interestingly, in single-enzyme assays the class I flavin reductases that contain flavin as a bound cofactor typically follow a ping-pong mechanism [26,27]. There was no observable change in the steady-state kinetic parameters of SsuE with varying ratios of the SsuD enzyme, however the kinetic mechanism of SsuE was altered to an equilibrium ordered mechanism in the presence of SsuD and saturating levels of octanesulfonate. With this mechanism the NADPH substrate and NADP+ product are in equilibrium with free enzyme. At saturating levels of FMN, the rate of the reaction will not be dependent on the concentration of NADPH, and the equilibrium is displaced toward the ternary complex. These results suggest that SsuD and the octanesulfonate substrate ensure that the reaction is driven in the forward direction even in the presence of low concentrations of NADPH. Thus, there is no Km value determined from the fit of the data for NADPH. Interestingly, the Km value for FMN determined from the plot of the initial reaction rates was increased 10-fold over the Km value for SsuE in the single-enzyme assay (Table 1). There are several reports of observed changes in the kinetic parameters for the two-component monooxygenase family of enzymes when comparing single-enzyme flavin reductase assays to luciferase-coupled assays. The class I flavin reductases FRP and FRG of bacterial luciferase have been shown to follow a ping-pong mechanism when monitoring single-enzyme reductase activity [22,23]. The class I flavin reductases contain a bound FMN cofactor that is reduced by NADPH, oxidized NADP+ is released, and a second flavin binds and is reduced by the bound flavin cofactor. The reduced flavin product in the absence of luciferase is oxidized in an unproductive dark reaction. Alternatively, in the coupled luminescence reaction measuring light emission, that includes luciferase and the decanal substrate in the reaction, the ping-pong mechanism is altered to a sequential mechanism. In addition, the Km values for FMN and NADPH are decreased significantly in the coupled luminescence reaction when compared to the single-enzyme assay. In the coupled luminescence reaction, the luciferase enzyme preferentially utilizes the FMNH2 cofactor and directly competes with the FMN substrate in reacting with the reduced flavin cofactor. The change in the steady-state kinetic parame-

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ters and mechanism strongly supports a model involving direct flavin transfer from the reductase to the luciferase enzyme. Conversely, there was no change in the steadystate kinetic parameters of the 4-hydroxyphenylacetate 3-monooxygenase system when monitoring the activity of the monooxygenase component in a coupled assay [24]. Instead, the mechanism of flavin transfer was proposed to occur through a diffusion mechanism due to the high affinity of the monooxygenase enzyme for reduced flavin. Based on previous data and our current analysis, there are several possible explanations for the observed changes in the steady-state kinetic parameters and value of mechanism for SsuE. The Km 0.016 ± 0.01 lM for flavin binding to SsuE in the single-enzyme assays is nearly equal to the Kd value, suggesting that the Km value essentially represents the dissociation constant. Therefore, the 10-fold increase in the Km value for SsuE in the presence of SsuD and the alkanesulfonate substrate can be attributed to a decrease in the binding affinity for oxidized flavin. Because flavin transfer between proteins is an essential step in catalysis, a lower affinity for oxidized or reduced FMN by SsuE would likely increase the rate of reduced flavin transfer to SsuD. In addition, a change to a rapid equilibrium ordered mechanism displaces the reaction towards the ternary complex and subsequent flavin transfer. The change in mechanism and observed increase in the Km value for the oxidized flavin with SsuE in the presence of SsuD and the alkanesulfonate substrate ensures that the critical flavin transfer step in the pathway is preserved. Therefore, flavin reduction by SsuE would lead to desulfonation of the alkanesulfonate substrate by SsuD as opposed to a nonproductive reaction leading to the generation of oxygen radicals. An alternative explanation for the differences in the Km values and steady-state mechanism is that there are two flavin binding sites in SsuE similar to those observed for bacterial luciferase FRG and FRP. The first site would result in nonproductive flavin reduction while the second site provides the reduced flavin utilized by SsuD. Although there is no bound flavin cofactor in the class II flavin reductases, this does not exclude the existence of a second flavin binding site. One could envision that in the absence of SsuD and the octanesulfonate substrate the flavin binds to SsuE at the nonproductive FMN binding site that possesses a higher affinity for oxidized flavin. However, during catalytic turnover with SsuD the flavin binds to the second binding site with the higher Km value for oxidized flavin. The reduced flavin located at the second binding site would be the preferred flavin substrate transferred to SsuD similar to the flavin transfer mechanism of bacterial luciferase. This may also explain why inhibition of SsuE at FMN concentrations above 0.2 lM was observed in the experiments with SsuD and octanesulfonate. The excess flavin may

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be binding to the nonproductive binding site and competing with SsuD in reacting with the reduced flavin involved in flavin transfer. The observed kinetic change to a rapid equilibrium ordered mechanism would correspond with the second flavin binding site. The necessity for the two binding sites in the bacterial luciferase FMN reductases has not been fully defined, and further experiments will be performed to explore the possibility of two flavin binding sites in SsuE. The results are the first to define an altered kinetic mechanism in the class II FMN reductases while solely monitoring flavin reduction. There are still many questions to be addressed regarding the mechanism of reduced flavin transfer in this family of enzymes, however our work provides a basis for future studies aimed at evaluating the mechanism of flavin transfer between the alkanesulfonate monooxygenase enzymes.

Acknowledgments The authors thank Dr. Douglas Goodwin for helpful discussions of the manuscript. We thank Dr. James C. Hu from Texas A&M University for his generous gift of E. coli strain K12. DNA sequencing was carried out at the Davis Sequencing Facility by Eric Bowman and Carrie Stoltz

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